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GENERATION OF A POTENT CHIMERIC TOXIN BY REPLACEMENT OF DOMAIN-III OF PSEUDOMONAS EXOTOXIN WITH RICIN-A CHAIN KDEL
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UNSPECIFIED (1995) GENERATION OF A POTENT CHIMERIC TOXIN BY REPLACEMENT OF DOMAIN-III OF PSEUDOMONAS EXOTOXIN WITH RICIN-A CHAIN KDEL. BIOCONJUGATE CHEMISTRY, 6 (5). pp. 624-629. ISSN 1043-1802.
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Abstract
Following cellular uptake, Pseudomonas exotoxin (PE) is cleaved by cellular protease which generates an enzymatically active C-terminal fragment (amino acids 280-613). This 37 kD fragment translocates to the cell cytosol where it ADP-ribosylates elongation factor 2 and inhibits protein synthesis. A recombinant hybrid toxin (designated PE-RTA) in which the ADP-ribosylation domain (domain III) was replaced by the RNA N-glycosidase domain of ricin (the A chain or RTA) has been produced in E. coli. The hybrid toxin effectively and specifically depurinated 28S ribosomal. RNA, indicating that the ricin A moiety folded into its native conformation. The cytotoxicity of PE-RTA for L929 cells was approximately 100-fold less than either native PE or whole ricin. However, the addition of the tetrapeptide KDEL to the C-terminus of PE-RTA (producing PE-RTA KDEL) increased cytotoxicity to the level of the native toxins. By analogy to PE, both PE-RTA and PE-RTA KDEL would be proteolytically cleaved within PE domain II during cell entry. A single amino acid substitution, believed to disrupt an essential step in the transport of the catalytically active PE fragment to the cell cytosol (Trp281 to Ala: Zdanovsky, A. G., Chiron, M., Pastan, I., and FitzGerald, D. J. (1993) J. Biol. Chem. 268, 21791-21799), reduced the cytotoxicities of both PE and PE-RTA KDEL by approximately 100-fold. Taken together, these data show that the ricin A chain component of the hybrid toxin requires essential PE-derived sequences at both the N- and C-termini of the translocating fragment. Clearly, in the context of this fusion protein, ricin A chain cannot effect its own transfer to the cytosol.
Item Type: | Journal Article | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Journal or Publication Title: | BIOCONJUGATE CHEMISTRY | ||||
Publisher: | AMER CHEMICAL SOC | ||||
ISSN: | 1043-1802 | ||||
Official Date: | September 1995 | ||||
Dates: |
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Volume: | 6 | ||||
Number: | 5 | ||||
Number of Pages: | 6 | ||||
Page Range: | pp. 624-629 | ||||
Publication Status: | Published |
Data sourced from Thomson Reuters' Web of Knowledge
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